JPH02490A - Production of monoclonal antibody - Google Patents

Abstract

PURPOSE:To enable utilization for elucidation of carcinogenic mechanism, diagnosis and treatment by culturing a hybridoma prepared by fusing a myelomatous cell to a plasmacyte of an animal suffering from autoimmune diseases in a culture medium. CONSTITUTION:The spleen of a mouse suffering from autoimmune disease, such as NZB, is extracted and treated to provide (A) a plasmacyte, such as splenic cell, which is then mixed with (B) a mouse myelomatous cell (e.g., NS-1) at (1-10):1 ratio. A fusion accelerator, such as polyethylene glycol, is added to carry out cell fusion and afford (C) a hybridoma, which is then cloned by a limiting dilution method, etc., to provide (D) a monocloned hybridoma. The resultant hybridoma (D) is subsequently cultured in a culture medium containing bovine fetal blood serum, etc., to afford a culture. The obtained culture is subsequently purified by an ammonium sulfate precipitation, gel filtration, etc., to produce a monoclonal antibody reactive with at least N-glypolylGM2 ganglioside.

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing a monoclonal antibody that reacts with at least N-glycolyl GM and ganglioside using a hybridoma. [Conventional technology] Glycolipids are constituent components of cell membranes, and the type, number, and
A variety of molecular species exist due to differences in binding methods, and exhibit species-specific, organ-specific, and cell-specific distributions. Its functions include acting as a receptor for bacterial toxins, hormones, etc., acting as an immunological determinant for blood group substances, and playing an important role in controlling proliferation or differentiation and interactions between cells. This is becoming clear. Furthermore, it has been shown that qualitative and quantitative compositional changes occur as cells become cancerous, and some of them may become cancer antigens, and that certain glycolipids are involved in the regulation of cell proliferation mechanisms mediated by growth factors and protein kinases. It has also been suggested that changes in its composition may be directly involved in the canceration mechanism, such as when it acts as a cancer. On the other hand, Milstein et al. reported a method for establishing a cell line that produces uniform antibodies with specificity for one type of antigenic determinant (Fuichicha; Nature. 256495-497, 1975), and a method for the qualitative determination of trace substances. ,
Quantification became possible. Using this technology to search for cancer antigens and differentiation antigens, many monoclonal antibodies specific to cancer cells and differentiating fetal cells have been produced.
It is clear that some of these antibodies are antibodies that recognize sugar chains of glycolipids or glycoproteins (Journal of National Cancer Institute; J, Na11. Cancerst, 7).
1231-251, 1983). For example, as monoclonal antibodies against human melanoma, antibodies that react with glycolipids such as GO, cangliondo, GD, and ganglioside have been obtained. In addition, monoclonal antibody N519-9 specific for pancreatic cancer reacts with glycolipids and glycoproteins having Nallon Lewis type A sugar chains. These antibodies are useful for diagnosing cancer and observing the progress of treatment, and further attempts are being made to use them for therapy. Qualitative and quantitative changes in glycolipids associated with canceration are caused by changes in the activities of various glycosyltransferases in the sugar chain biosynthesis mechanism due to abnormal gene expression. A sugar chain structure that does not occur is created. That a! The f-chain structure can be used as a cancer marker. As described above, the importance and usefulness of glycolipids as clues to elucidate the canceration mechanism and as cancer antigens and cancer markers are attracting attention, and their application in clinical fields such as diagnosis and treatment is expected. . Among glycolipids, those containing sialic acid, which is an acidic sugar, in their sugar chains are collectively called gangliosides. The main types of sialic acid are N-acetylneuraminic acid and N-glycolylneuraminic acid. Sialic acid is widely detected in various organs, cells, and body fluids of animal species, but N-glycolylneuraminic acid has so far not been found in normal humans or chickens. In the following description, when it is necessary to specifically distinguish the types of these sialic acids, N-glycolyl or N-acetyl is added before the name of the glycolipid to distinguish them. Serum sickness, [I: Heterophilic antibodies detected and agglutinating red blood cells of sheep, horses, pigs, rabbits, and guinea pigs include Hanganatziu Deich.
er, hereinafter abbreviated as 11-D) antibody. The antigen identified by this antibody is called HD antigen. N-glycolylneuraminic acid-containing ganglioside is H
-D antigen activity [Biochemical and Biophysical Research Communications (Biochem, Biophys. Res, Commun,) Vol. 79, No. 388-39
5 (1977)), the sugar chain structure of NeuGcσ2-3Gal was identified as the main antigenic determinant. In recent years, N-glycolyl GM, a ganglioside with )ID antigen activity, ganglioside (U3NeuG
immunize chickens with c-LacCer) and extract H from the serum.
Antibodies that react with various gangliosides having -D antigen activity were produced, and using these antibodies, it was reported that N-glycolylneuraminic acid is characteristically present in human tissue [Viken Journal ( Biken J, ), 2nd
5, pp. 47-50 (1982) 1. In addition, several types of HD antigen-active h-glycolylneuraminic acid-containing gangliosides were detected in glycolipids extracted from human colon cancer tissues. In teratoma tissues, glycoproteins with sugar chains active against HD antigen were detected [Gann et al.
), Vol. 75, pp. 1025-1029 (1984)
]. When glycolipids with HD antigen activity detected from human colon cancer tissues were analyzed using antibodies produced in chicken, N-glycolyl GM, gaglioside, N-glycolyl GM3 ganglioside, 0-acyl-N- Glycolyl GM3 ganglioside, IV 3NeuGc-nLc
ose+Cer, and these were not detected in normal tissue [Cap Research (Cancer Res.
), Vol. 45, pp. 3796-3802 (1985) 1. Gangliosides with ToD antigen activity were also detected in cell lines of Marek's disease, a type of chicken lymphoma [Journal.・Of Biochemistry (Tokyo); J, Biochem, (Tokyo), No. 9
5, pp. 785-794 (1984)]. ] N-glycolylneuraminic acid N-glycolyl GM,
N-glycolylneuraminic acid-containing sugar chains, including ganglioside sugar chains, are considered cancer-related antigens, and detecting these with high sensitivity and accuracy is extremely important for cancer diagnosis. N-glycolylneuraminic acid or N-glycolyl GM2
In order to efficiently detect N-glycolylneuraminic acid-containing sugar chains, including ganglion sugar chains, immunoassay methods are considered to be superior in terms of detection sensitivity and accuracy. Antibodies that react with N-glycolylneuraminic acid-containing sugar chains that have HD antigenic activity have traditionally been obtained by immunizing chickens with purified glycolipid antigens that have HD antigenic activity and separating them from their serum. [Molec, Jmmunol, Vol. 19, No. 87-
94 pages (1982)]. ]N-Glycolyl GM! Polyclonal antibodies with high specificity for gangliopsis are injected into chickens with N-glycolyl GM,
Gangliosides can be obtained by immunization [Biochemical and Biophysical Research Communications (Biochem. Biophys, Res, Commun,), Vol.
Volume 29, pp. 334-341 (1985)],]N-
Glycolyl GM, ganglioside and N-glycolyl GM
Polyclonal antibodies reactive against N-glycolyl GM3 ganglioside can be obtained by immunizing chickens with N-glycolyl GM3 ganglioside [CancerRes, Vol. 45, No. 3796-3]
p. 802 Cl985)]. However, these methods have some drawbacks. That is, (1) in order to obtain antiserum, a large amount of purified antigen is required each time. (2) There are variations in affinity and titer mainly due to individual differences in immunized animals. (3) Since antibodies other than the target antibody are also present, complicated operations are required to purify the target antibody. (4) There is a drawback that there is a limit to the amount that can be produced at a time. Therefore, in order to perform immunological assays accurately and with maximum effectiveness, it has been desired to be able to supply large quantities of antibodies of stable and uniform quality that are free from contamination with other antibodies. Production of such antibodies has already been reported as a monoclonal antibody production technique. On the other hand, it has been reported that a human monoclonal antibody that specifically recognizes melanoma, which was obtained by turning the lymphocytes of melanoma patients into cancer with EB virus, reacts with GM2 ganglioside (Procedure
Observation of the National Power of Science of the USA; Proc. Natl, Acad, Sci, U, S, A...
80, 5392-5396゜1985). In addition, although GM2 ganglioside is almost absent in normal red blood cells, human erythroleukemia cells have
GM! It has been reported that gangliosides accumulate
(Blood; Blood, familiar, 1230-124
1, 1983). In addition, N
- Using monoclonal antibodies that recognize acety-11 GM2 ganglioside and N-glycolyl GM2 ganglioside, it has been shown that GM and ganglioside are mainly expressed in neuroectoderm cancer cells (Cathy Research; Cancer Res, 46 .4116~4
120.1986). In this way, N-acetyl GM! Gangliondo and N
- Glycolyl GM and ganglioside are considered cancer-related antigens, and detecting them with high sensitivity and accuracy is important for cancer diagnosis. As mentioned above, N-glycol GM, gangliosides, their related glycolipids, and their sugar chain structures are extremely important as cancer-related antigens, and therefore N-glycolyl GM2
Monoclonal antibodies with reactivity with gangliosides, such as N-glycolyl GM! Gangliosides and N
- Monoclonal antibodies that have specific reactivity with acetyl GIJ and gangliosides, and monoclonal antibodies that have specific reactivity with N-glycolyl GM2 ganglioside and other glycolipids containing N-glycolylneuraminic acid, It is extremely useful for diagnosing various diseases, especially human cancer diagnosis by tissue, cell diagnosis, blood, urine diagnosis, image diagnosis, etc. In addition, it can be applied to missile therapy that binds drugs to antibodies and treatments that utilize cytotoxicity, and it can also be applied to diagnosis by detecting antibodies against sugar chains. In addition, various monoclonal antibodies that are reactive with N-glycolyl GM and gangliosides are used for basic research on the relationship between sugar chains and canceration mechanisms, the structures of sugar chains and glycolipids, and their roles and functions in vivo. It can be used as a useful tool. Thus, it is considered extremely important to produce a more/clonal antibody that recognizes at least N-glycolyl GM2 ganglioside. However, N-glycolylneuraminic acid is present in many animals including mice except humans and chickens, and N-glycolylneuraminic acid, which is the subject of the present invention,
N-glycolylneuraminic acid-containing glycolipids, including gangliosides, are known to be widely present in mouse tissues, and these glycolipids are considered to be self-antigens for mice and have extremely weak immunogenicity. Therefore, Ba1b
In conventional methods using normal mice such as /c mice as immunized animals, it has been extremely difficult to obtain monoclonal antibody-producing hybridomas against N-glycolylneuraminic acid-containing glycolipids, including N-glyfuryl GM and gangliosides. On the other hand, animals with autoimmune diseases are known to produce antibodies against self-antigens such as anti-nuclear antibodies and anti-erythrocyte antibodies [Immunological Review (Ia+5unol. Rev.), Vol. 55, pp. 121-154. (1981
Year)〕. By immunizing animals with autoimmune diseases, especially mice with autoimmune diseases, the present inventors were able to increase the immune reactivity to N-glycolylneuraminic acid-containing glycolipids, which are thought to be self-antigens, and to create hybridomas. I thought it was possible, so I tried it and achieved my goal. In this process, it was discovered that hybridomas that produce monoclonal antibodies that recognize N-glycolyl GM and gangliosides can be easily produced, leading to the completion of the present invention. [Object of the Invention] An object of the present invention is to provide a method for producing a monoclonal antibody that specifically recognizes at least N-glycolyl GM2 ganglioside. [Structure of the Invention] The present invention relates to a method for producing a monoclonal antibody that recognizes at least N-glycolyl CM and ganglioside, and is characterized in that it is produced by a hybridoma obtained by fusing myeloma cells and plasma cells of an autoimmune animal. do. The structures of the glycolipids described hereinafter are shown below. N-acetyl GM, ganglioside (U'NeuAc
-GgOse4 Cer)Galβl-"3GalNA
cβI-”4Galβl”4G1cβl-1cer↑ 2 a NeuAc N-glycolyl GM, ganglioside (II 3Neu
Gc-GgOse, Cer)Gal /j 1-”
3GalNAcβl-4Galβ1-4Glcβl→l
Cer2a NeuGc N-glycolyl GM, ganglioside (M3NeuG
c-GgOse, Cer)GalNAcβ1-4G
al fi l→4G1cβ1-ICer↑ 2 a NeuGc N-glycolyl GM, ganglioside (If' Ne
uGc-Lac Cer)Galβl-4Glcβl-
*ICer↑ 2 a NeuGc TV 3NeuGc-nLeose4CerGa 17
? 1-4G IcNAcβ1-3Galβl-4GI
cβl-1cerN-acetyl GM2 ganglioside (
II 3NeuAc-GgOse, Cer)Gal
NAcβI→4GalβI→4GIcβI-ICer2
a NeuAc N-acetyl GM, ganglioside (II 3NeuA
c-LacCer)Galβ1-”4Glcβ1=1
cer↑ 2a NeuAc GD, ganglioside (II 3(NeuAc), -G
gOse, Car) GalNAcβ1--40alβ
l-4Glcβl+1Cer↑ 2 a NeuAc ↑ 2 a NeuAc Asialo GM, (GgOse, Cer)GalNAc
β1→4Galβl→4Glcβl−=ICerCD
I (Lac Cer) Galβl→4G1cβl-"1cerIn the formula, Gal is galactose, Glc is glycose, and Ga1NAc is N
- Acetylgalactosamine, GIcNAc means N-acetylglucosamine, NeuGc means N-glycolylneuraminic acid, NeuAc means N-acetylneuraminic acid, and Cer means ceramide. A method for producing a hybridoma that produces the monoclonal antibody will be explained below. Autoimmune disease animals are used as mammals for obtaining plasma cells, and they are preferably selected in consideration of compatibility with the myeloid cells used for cell fusion, with mice and rats being preferred. The autoimmune disease mouse that can be used is NZB. NZW, B/WFI, MRL/1. BXSB male, S
Examples include L/Nl mice of each strain, and examples of autoimmune disease rats include spontaneously hypertensive rats. In addition, polyclonal B cell activators (PBA) such as Durham-negative bacterial lipid polysaccharide (LPS) and dextran sulfate
Ba whose autoantibody production ability was increased by administering
Normal mice such as 1b/c may be brought into an autoimmune disease state and used. As an immunogen, it is possible to use any of the target N-glycolyl G ganglioside-containing cells themselves, cell membrane components separated from the cells, and N-glycolyl GM and gangliosides separated from the cells.
It is also possible to use glycolipids together with phospholipids and cholesterol in liposomes. Immunization is carried out by a general method, and the above immunogen is added to a phosphate buffer solution (hereinafter referred to as PBS).
The drug may be diluted with, for example, and administered intraperitoneally or intravenously. At this time, the immunogen may be supported on a carrier such as bovine serum albumin (BSA) or bacterial cells, or an adjuvant such as 70-indoor adjuvant or bacterial cell adjuvant may be injected together. Furthermore, the method of the present invention using mice with autoimmune disease does not necessarily require the use of an immunogen. N-glycolyl GM for mouse red blood cells! The existence of gangliosides [Journal of Biochemistry (
Tokyo) (J, B 1ocbe+s, (Tokyo)
yo)), Volume 91, Pages 1039-1046 (198
2 years)], and autoimmune disease mice produce autoantibodies including anti-erythrocyte antibodies, so autoimmune disease mice have N.
-Glycoryl GM! It is thought that antibodies against gangliosides are produced, or are likely to be produced, as anti-erythrocyte autoantibodies or other autoantibodies. Therefore, when using autoimmune disease mice, it is possible to create monoclonal antibody-producing hybridomas that react with N-glycolyl GMz ganglioside without using an immunogen. If no immunogen is used, there is no need to inject any autoimmune disease mice, but various adjuvants such as 70 India complete adjuvant, 70 India incomplete adjuvant, killed pertussis cells, killed salmonella cells, etc. It is preferable to increase the immune response capacity of mice with autoimmune disease by injecting them. Plasma cells taken from the animal are fused with myeloma cells. Plasma cells are commonly obtained as lung cells. The myeloma cells are preferably mouse myeloma cells, and various known cells such as NS-1%SP-2, X
63.6.5.3, P3-Ul, etc. can be used. The fusion method is performed according to a known method. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ), etc. are used. The ratio of lung cells to myeloma cells used is the same as in the general method, 1:1-10.
Pair 1 is preferred. After completion of fusion, hybridomas are selected by culturing in a normal selection medium. The myeloma cells described above are HA
Since cells cannot grow in T medium (a medium containing hypoxanthine, aminopterin, and thymidine), cells that grow in HAT medium may be selected. When the hybridoma colony becomes sufficiently large, a strain producing the desired antibody is searched for and cloned. The search for the antibody-producing strain can be carried out using methods generally used to detect antibodies, such as the ELISA method (Method in Enzymology; Meth, Enzymol, 70).
．． 419-439.1980), agglutination reaction method, RrA method, double immunodiffusion method, etc. Specifically, a plate to which a purified glycolipid antigen has been attached is blocked with BS^, and then reacted with the culture supernatant of a test hybridoma, and further reacted with an enzyme-labeled antibody against a mouse antibody, and the antigen The presence of the antibody bound to the antibody is confirmed by measuring enzyme activity, and the desired antibody-producing strain is selected. Further, cloning is performed by the limiting dilution method. That is, on a 96-well microtiter plate, hybridomas are distributed so that one or less per well is grown to grow a single colony. At this time, it is preferable to add mouse thymocytes as feeder cells. The above cloning is repeated to obtain monocloned hybridomas. Monoclonal antibodies produced by hybridomas can be obtained by culturing hybridomas in a medium and separating them from the culture supernatant, or by intraperitoneally administering hybridomas to mice and collecting them from the ascites fluid. Furthermore, −
It is also possible to purify using intensive methods, such as ammonium sulfate precipitation, gel filtration, ion exchange column chromatography, etc. [Examples] Hereinafter, the present invention will be explained in more detail with reference to Examples, but these Examples are not intended to limit the scope of the present invention in any way. Example 1 1) Isolation and purification of antigens and various glycolipids ■ Rabbit thymus cell membrane components Thymus tissues obtained from two 6-week-old rabbits were homogenized in PBS and centrifuged at 800 rpm for 5 minutes. The upper groove was centrifuged at 10,000 g for 1 hour, and the resulting pellet was dispersed in PBS lO+a (2) and used as an immunogen. was added, and from the resulting precipitate, the volume ratio of chloroform:methanol:water (10:20:1) (10:
Extraction operations were performed sequentially with each mixed solution of 10:0) C20:10:1). The obtained crude glycolipids were treated with DEA.
The mixture was subjected to E-Sephadex A-25 column chromatography to separate into acidic and neutral fractions, and the acidic fraction was subjected to Iatrobeads column chromatography to obtain N-glycolyl GM and ganglioside. Similarly, N-glycolyl GM and ganglioside were obtained from horse erythrocytes, IV 3NeuGc-nLcose, Cer from bovine erythrocytes, and N-acetyl GM from the cerebrum! Ganglioside and N-acetyl GM3 ganglioside were obtained from human red blood cells as CDI. N-acetyl GM2 ganglioside was reacted in IN formic acid at 100°C for 1 hour, and subjected to DEAE-Sephadex A-25 column chromatography and then Iatrobeads column chromatography to produce Asialo GM! I got it. Glycolipid-containing liposomes for immunosensitization are glycolipid ll11
g, phosphatidylcholine 411g, cholesterol 1
After completely removing the solvent from a 0 mg mixed solution of chloroform:methanol (1:1) in a flask under reduced pressure, PBS511ff was added thereto, and the mixture was subjected to ultrasonication. 2) Immunization method and cell fusion (1) First rabbit thymus cell membrane component PBS solution and the same amount of Freund's complete adjuvant were emulsified and mixed, and 400 μa was injected intraperitoneally into a NZB mouse (female, 6 weeks old). Two weeks later, using an additional 70 int incomplete adjuvant,
Injected in the same way. 13 weeks later, TV 3NeuGc-n
LcOse, Cer-containing lipome PBS solution 100.
uQ was injected intraperitoneally, and two weeks later, liposomes were injected in the same manner. Cell fusion Three days after the final immunization, the lungs were removed from the mice, disaggregated into single cells, and the tile cells were washed with RPM11640 medium. On the other hand, mouse myeloma cells X63 in logarithmic growth phase
．． 6.5.3 was collected and washed with RPM [1640 medium. A suspension of 7 x 10' tile cells and a suspension of 1.4 x 10' mouse myeloma were mixed, and the medium was removed by centrifugation. In a water bath heated to 37°C, 1 ml of 50% polyethylene glycol RP old 1640 medium was gradually added to the mixed cells over 1 minute, and fusion was performed by stirring gently for 1 minute. RP old 1640 medium 2ea (l
was added for 2 minutes, and then 7 mQ was added for 2 minutes with gentle stirring. Remove the medium by centrifugation and add l
RPM11640 medium 20IIIQ containing 0% tallow serum
was added and distributed to two 96-well plates in an amount of 0.1 m12 per hole. The next day, add HAT medium (4X 10-'M aminopuritene, 1.6x 10-'M thymidine, lX
R containing 10-4M hypoxanthine, 10% tallow serum
PM11640 medium) 0-111112 was added to each well. The medium in each well was further added to HAT every 3 or 4 days.
Half of the volume was replaced with medium. After 3 weeks, hybridoma growth was observed in 80% of the wells. (2) Second time Rabbit thymus cell membrane component PBS solution and the same amount of 70 India complete adjuvant were emulsified and mixed, and 300 μQ was injected intraperitoneally into a NZB mouse (female, 6 weeks old). Thereafter, N-glycolyl GM and 20 μg of ganglioside PB were adsorbed on 80 μg of acid-treated Salmo laminate bacteria.
200 μQ of S solution was intravenously injected 5 times every 2 weeks. Three days after the final immunization, the lungs were removed from the mice, disaggregated into single cells, and the tile cells were washed with RPMI1640 medium. On the other hand, mouse myeloma cells X63 in logarithmic growth phase
．． 6.5.3 was collected and washed with RPM11640 medium. A suspension of 4.7 x 10" tile cells and a suspension of 9.2 x 10" mouse myeloma were mixed and the medium was removed by centrifugation.The mixture was heated to 37°C and mixed in a water bath. cells with 50% polyethylene glycol-RPM11.
640 medium 2 + w (2 was gradually added over 1 minute, and fusion was performed by stirring gently for 1 minute. RPM 4 mQ of 640 medium was added over 2 minutes, and then 14 m < 1 was added over 2 minutes with gentle stirring. The medium was removed by centrifugation, 120 m<+ of RPM11640 medium containing 10% tallow serum was added to the cells, and the mixture was distributed into 12 96-well plates at 0.1 m2 per well.The next day, HAT medium (4X
0.1 tQ of RPM11640 medium containing 10-'M aminopritene, 1.6 x 10-'M thymidine, l x 10-'M hypoxanthine, 10% tallow serum) was added to each well. The medium in each well was further increased for 3 or 4 days.
Half of the volume was replaced with LAT medium (every day). After 3 weeks, 90% growth of well hybridomas was observed. ■The third immunization method was carried out in the same manner as the second one, except that intravenous injections were performed eight times. Cell fusion: 3.6 x 10' tile cells, mouse minirow? 7. Using 1XlO' cells, add 100 mQ of RPM r medium containing lθ% beef tallow serum, and perform a 96-well plate 1-1.
The process was the same as the second time except that 0 sheets were distributed. After 3 weeks, hybridoma growth was observed in 80% of the wells. 3) Selection of hybridomas Antibodies in the cultured grooves of hybridomas were searched for by ELISA method. As antigens, N-glycolyl 0M2 ganglioside, N
-Glycolyl GM3 ganglioside and IV3NeuG
c-nLcOse, Cer was used. 500ng of each antigen
was adsorbed onto a microtiter plate for ELISA, and
After blocking with % BSA-PBS solution, the culture supernatant was reacted. Furthermore, the antibody of interest was detected by reacting with a peroxidase-labeled goat anti-mouse immunoglobulin antibody and measuring the absorbance at 492 nm using orthophenylenediamine as a substrate. the result,
In the first immunization, activity was detected in one well of the hybridoma produced by cell fusion, and in the second immunization, activity was detected in one well.
Activity was detected in two wells of the hybridomas produced by cell fusion. In addition, activity was detected in four wells of the hybridomas produced in the third immunization and cell fusion. These hybridomas were transferred from HAT medium to HT medium without aminopriten, and further supplemented with 10% tallow serum (F).
CS)-containing RPM 640 medium and cultured. Example 2 Measurement of antigen specificity of each monoclonal antibody by ELISA method: ELISA method was performed using various glycolipid Q and 2n+sol as antigens. The antigen adsorbed on the plate was reacted with the hybridoma culture supernatant, and further reacted with a peroxidase-labeled goat anti-mouse immunoglobulin antibody. Using orthophenylenediamine as a substrate, the absorbance of 492n11 was measured to examine the reactivity of monoclonal antibodies to various antigens. The results are shown in the table below. ＋ ＋ Among the monoclonal antibodies obtained, PYK-2, YH
D-04 and YHD-07 are N-glycolyl GM! Showing strong reactivity to gangliosides, YHD-02, Y
HD-03 and YHD-05 also showed reactivity with N-glycolyl GM, ganglioside, and 1NeuGL-nLcOse and Cer in addition to N-glycolyl GM and ganglioside. In addition, YHD-06 is N-glycolyl GM,
The reaction of ganglioside with N-acetyl CM2 ganglioside is shown. That is, PyK-2, YHD-04, YHD-06 and YHD-07 were used as immunogens.
No reactivity was shown for c-nLcose, cer or N-glycolyl GM3 ganglioside, and N-glycolyl G
M! showed strong reactivity towards gangliosides. Example 3 Examination of glycolipids in rabbit thymus tissue on a thin layer chromatography (hereinafter referred to as rTLcJ) plate: The rabbit thymus tissue used as an immunogen in Example 1 contains N-glycolyl GM, ganglioside, and rV3NeuGc- nLc
It has been reported that there are many N-glycolylneuraminic acid-containing glycolipids such as ose4cer [Biochim, B
iophys, Acta), Vol. 665, No. 205-2
13 (1981)], but the existence of toglycolyl GM and gangliosides is unknown. We attempted to confirm this using monoclonal antibodies. For rabbit thymus tissue, the volume ratio of two bottles of chloroform:methanol (10:20:l), (10:10:O),
(20:10:l) extraction operation was performed in sequence with each mixed solution, and the obtained crude glycolipids were subjected to DEAE-Sephadex-25 column chromatography to obtain an acidic fraction containing gangliosides. For the acidic fraction obtained,
TLC and enzyme immunostaining were performed using monoclonal antibody PyK-2. 6111 at 1CI11 from the bottom edge of the TLC plate.
The sample was sporatiged with a width of 111, and the solvent system chloroform: methanol + 2.5N aqueous ammonia (55:4
5:10 volume ratio). Of the two plates subjected to the same operation, one was developed with orcinol reagent. The other plate was reacted with PyK-2 and further reacted with a peroxidase-labeled goat anti-mouse immunoglobulin antibody. Using 4-chloro-1-naphthol as a μ substance, a bluish-purple colored spot was detected. The results are shown in the figure. A is a plate subjected to color development using an orcinol reagent, and B is a plate subjected to enzyme immunostaining. 1 and 3 each contain 2 nmol 12.30 p of N-glycolyl GM2 ganglioside.
mol sposo h I,, 2.4 has sial dissociation ffi
The acidic fraction of rabbit thymus tissue chloroform:methanol extraction of 2 n1lloI was spotted and developed. It was confirmed in this example that N-glycolyl GM2 ganglioside does not exist in the rabbit thymus tissue used as an immunogen in Example 1 of the present invention. It was confirmed that hybridomas producing reactive antibodies were created without injecting N-glycolyl GM2 ganglioside into NZB mice. Example 4 1) Measurement of antigen specificity of YHD-06 by ELISA Using various glycolipids as antigens, the antigen was adsorbed in stepwise amounts onto an ELISA plate, and the /S hybridoma culture supernatant was Made it react. After washing with PBS,
After reacting with a peroxidase-labeled goat anti-mouse immunoglobulin antibody and washing with PBS, the absorbance at 492 μm was measured using orthophenylenediamine as a substrate to examine the reactivity of the antibody. The results are shown in FIG. In the figure, the vertical axis represents the absorbance at 492 cm, and the horizontal axis represents the amount of antigen. In addition, each symbol indicates the following. White Maruni N-glycolyl GM2 ganglioside (U'
NeuGc-GgOse, Cer) white triangular di-N-acetyl GM2 gangliondo (n 1NeuAc-Gg
Ose3 Cer) White square: N-acetyl GM, ganglioside (U3NeuAc-GgOse, Cer)
:N-Glyph 9110M. Ganglioside (II 1N
euGc-GgOse4 Cer): N-acetyl GM
, ganglioside (I[3NeuAc-Lac Cer) diN-glycolyl GM, ganglioside (I[''NeuGc-La
c Cer): GD, ganglioside (U3(NeuAc), -GgOses Cer) Nearsia oGM2 (GgOse3 Cer): CDH(
Lac Car) As is clear from Figure 2, antibody YHD-06 is N-glycolyl GM! Gangliosides and N-acetyl GIJ
It was confirmed that it has strong compatibility with 2 gangliosides and does not react with other gangliosides or asialoglycolipids. 2) YHD-0 on thin layer chromatography plate
Measurement of reactivity of step 6 Various glycolipids were spotted at lc+s from the lower end of the TLC plate using a 6-point radius, and developed with an appropriate solvent system. Of the two plates subjected to the same procedure, one was developed with orcinol reagent I; The other piece was subjected to enzyme immunostaining. That is, the antibody of the present invention was reacted, and then a peroxidase-labeled goat anti-mouse immunoglobulin antibody was reacted. Using 4-chloro-1-naphthol as a substrate, a bluish-purple colored spot was detected. FIG. 3 shows the results using various glycolipids as antigens. Chloroform as developing solvent: methanol: 2.5N
Aqueous ammonia (55:45:10 volume ratio) was used. A
1 is a plate subjected to color development using Or-Nol reagent, and B is a plate subjected to enzyme immunostaining. ■N-acetyl CM,
Ganglioside, N-acetyl 0 M2 ganglio/do and N-acetyl GM, ganglioside, 2 contains N-glycolyl GM, gangliondo, N-glycolyl GM2 gangliondo and N-glycolyl GM3 ganglioside, 3 contains asialo GM, and CDH Expanded. Apparently, the antibodies of the invention are N-glycolyl GM2 ganglioside and N-acetyl GM! It can be seen that it reacts only with gangliosides.

【Effect of the invention】

According to the present invention, various monoclonal antibodies having reactivity with N-glycolyl GM and ganglia can be provided. This antibody is very effective for elucidating the mechanism of cancer development, diagnosis, and treatment, and is also useful for basic research on glycolipids and glycoproteins.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the reactivity of monoclonal antibody PyK-2 with N-glycolyl GM, gangliosides, and the acidic fraction extracted from rabbit thymus tissue in chloroform and methanol. FIG. 2 is a graph showing the reactivity of the antibody YHD-06 of the present invention to various glycolipids by changing the amount of antigen in the ELISA method. It is a spot diagram showing the reactivity with YHD-06 by enzyme immunostaining.

Claims (4)

[Claims] (1) A method for producing a monoclonal antibody, characterized in that a monoclonal antibody that reacts with at least N-glycolyl GM_2 ganglioside is produced by a hybridoma prepared by fusing myeloma cells with plasma cells of an animal with an autoimmune disease. (2) The method for producing a monoclonal antibody according to claim 1, wherein the myeloma cells are mouse myeloma cells. (3) The method for producing a monoclonal antibody according to claim 1 or 2, wherein the autoimmune disease animal is an autoimmune disease mouse. (4) Claim 3, wherein the autoimmune disease mouse is an autoimmune disease mouse that is not immunized with N-glycolyl GM_2 ganglioside, its sugar chain, or substances containing them.
Methods for producing the monoclonal antibodies described.